An increasing body of data suggest that aberrant DNA methylation of selected clusters of CpG dinucleotides ("CpG Islands") may act as a "mutation" which can silence gene expression and participate In chromosome changes critical to neoplastic progression. Also, increased cellular capacity to methylate DNA, via increases in DNA methyltransferase gene expression, is a very early change in experimental and native tumor progression models. Our proposal seeks to validate, further, interaction between the above events and their direct role in the evolution of human cancer. The molecular and cell phenotype consequences of two discrete CpG island methylation events are under study. We have Identified, through analysis of a hypermethylated CpG island at 17p13.3, a new gene with excellent features for being a candidate tumor suppressor residing telomeric to p53. The potential suppressor role and molecular function of this gene will be determined. Also, aberrant methylation of a 5' CpG island associated with the estrogen receptor (ER) gene on 6q has been found in virtually all samples of human and murine colon neoplasms and leukemias tested and also in cultured ER negative human breast cancer cells. The role of this change will be studied in the hematopoietic cell system. We have tightly linked loci of aberrant CpG island methylation, especially that above at 17p13.3, with timing and incidence of allelic losses for different types of human cancer (colon, brain, renal, lung). We will determine how the CpG Island changes might Influence chromosome function to predispose to structural abnormalities. Hypermethylated CpG Islands are known to be associated with delayed DNA replication timing on the Inactive X-chromosome, and at the fragile-X gene locus. We will determine whether such delays may be the case for domains around methylated CpG islands in neoplastic cells. Finally, we will expand our recent findings that overexpression of an exogenous DNA methyl transferase (DNA-MTase) gene causes hypermethylation and transformation of NIH 3T3 cells. We will attempt to use this maneuver to model selected human tumor progression steps. We will also evaluate the phenotypic consequences of general, and tissue specific, overexpresslon of an exogenous DNAMTase gene In transgenic mice.